Degeneration of the larval midgut of Bombix Mori during metamorphosis: role and regulation of autophagy and apoptosis.

The midgut of the silkworm, Bombyx mori, is extensively remodeled during metamorphosis: in fact, while cell death processes lead to the degeneration of the larval epithelium, the adult midgut is formed by the proliferation and differentiation of stem cells. Our group previously described the intervention of apoptotic and autophagic events in larval midgut cells undergoing degeneration (Franzetti et al., 2012). The present study aims at investigating the molecular pathways of apoptosis and autophagy, the role of the two processes and their relationship in this tissue. We first analyzed the expression pattern of autophagic and apoptotic genes, as well as used specific markers to assess the precise timing of autophagy and apoptosis during metamorphosis. The results obtained confirm that autophagy is activated at spinning stage, while apoptosis intervenes with a delay of 24-48 hours. The final demise of apoptotic cells occurs by secondary necrosis and their content is released in the lumen of the adult midgut. To investigate the mechanisms that lead to the activation of autophagy and apoptosis we used 20-hydroxyecdysone (20E), one of the main regulators of metamorphosis in insects. The administration of a single dose of 20E induces the transcription of both autophagic and apoptotic genes, but fully activates only autophagy. In contrast, the activation of effector caspases needs a second injection of 20E. These data suggest that, during development, autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with 20E metamorphic peak. Moreover, our results demonstrate that 20E activates autophagy by inhibiting the Tor pathway. However, inactivation of Torc1 through rapamycin administration is not sufficient to trigger and maintain a full autophagic response, thus suggesting that the activation of autophagy by 20E is mediated by multiple downstream targets. In order to study the role of autophagy and apoptosis in this setting we used specific inhibitors. The impairment of the autophagic flux, through administration of chloroquine, determines an increased degeneration of the larval midgut epithelium and higher levels of caspase activity compared to controls, while the inhibition of caspase activation, by using z.vad.fmk, leads to a severe delay in the degradation of the epithelium. These data demonstrate that, while autophagy has a pro-survival role in this setting, apoptosis is the major process that drives the demise of the larval midgut during metamorphosis.